Angiogenesis, the development of blood vessels from pre-existing vasculature, is critical in many diseases. Enhancement of angiogenesis is beneficial in cardiac and cerebral ischemia and wound healing, while anti-angiogenic strategies are being examined in cancer and retinopathies. Acting via adenosine receptors (AR), adenosine regulates angiogenesis as it is produced during hypoxia. A critical step in angiogenesis is endothelial cell (EC) proliferation and activation of extracellular signal regulated kinase (ERK1/2) is considered to be a key underlying event. Our studies show that the A2BAR elicits proliferation and ERK1/2 activation in human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMVEC). Furthermore, we found the recently discovered Epac1 protein is required for ERK1/2 stimulation in response to A2BAR activation. Epac1 is a guanine nucleotide exchange factor for RapGTPases and provides a basis for cAMP-dependent, Protein Kinase A independent responses. The goal of this proposal is to delineate the signaling pathway downstream of Epac1 that results in ERK1/2 activation and to determine if direct and A2BAR-mediated activation of Epac1 and downstream proteins results in EC proliferation and tube formation. Both HUVEC and HMVEC, collectively referred to as ECs will be used in all studies. In Specific Aim 1, it is hypothesized that Epac1 signals to ERK1/2 in ECs via a cascade involving RapGTPase(s) and B-Raf. This will be explored by a strategy employing Rap pull-down assays, expression of constitutively active and dominant negative Rap proteins and siRNA to knockdown expression of specific Rap isoforms and B-Raf. Additionally, the subcellular location of activation of key proteins will be analyzed. In Specific Aim 2, it is hypothesized that Epac1 and associated downstream proteins promote EC proliferation. We will determine the effects of a specific Epac1 activator and overexpression of Epac1 on proliferation. Additionally, siRNA and dominant negative constructs will be used to determine the necessity of Epac1 and associated proteins in A2BAR-mediated proliferation. In Specific Aim 3, the effects of Epac1 will be determined in endothelial tube formation, an in vitro model of angiogenesis. This proposal will further an understanding of Epac1 function in EC biology with a focus on the unexplored areas of proliferation and angiogenesis. PUBLIC HEALTH RELEVANCE: The development of new blood vessels may be beneficial or detrimental in different diseases. This development requires the growth and assembly of endothelial cells which form the inner lining of blood vessels. This project examines the role of a protein known as EPAC in growth and ordered assembly of these endothelial cells.